The overall objective of this SBIR proposal is to develop and commercialize a human 3D or mini- liver bioassay system suitable for real-time analysis of metabolic functions and long-term drug toxicity profiles. At present, liver functions are mostly studied using HepG2 cell line and primary hepatocytes cultured onto collagen or polymer matrices. However, these 2D monolayer models allow only partial or short-term toxicity analysis due to rapid loss of metabolic functions. Interestingly, spheroid formation in 3D matrix/scaffold cultures is shown to improve host cell functions. In Phase I studies, we successfully demonstrated the feasibility of a new human bioscaffold- 3D HuBiogel (high-density gel) for supporting long-term survival and maintenance of primary liver cells. Moreover, integration of rotary bioreactor system allowed efficient production/analysis of viable mini-liver spheroids. We now propose to complete the development of new 3D-liver bioassay platform that exhibits improved metabolic functions, and thus will enable precise drug toxicity testing in vitro. Phase II specific goals are: 1. Establish an efficient hepatic cells cultivation protocol by optimizing 3D HuBiogel scaffolds and rotary bioreactor or perfusion culture strategies; 2. Test functionality of 3D-liver culture model by evaluating key biochemical and metabolism endpoints; and 3. Validate the practical utility of mini-liver assay system via real- time analysis of CYP450 induction and chemical/drug toxicity responses. Comparative control studies will include 3D culture scaffolds of Type-I collagen or Matrigel as well as fresh human liver-slice cultures. High throughput adaptability of 3D-liver bioassay will also be examined for toxicogenomics via CYP-microarrays. Thus, biologic relevance of new marketable 3D-liver bioassay system will be confirmed for improved drug toxicity analysis/prediction in humans. We are confident this advanced in vitro hepatotoxicity model will positively impact basic, preclinical and biomedical research arenas. Commercial Importance & Significance: No acceptable commercial liver toxicity assay currently exists which utilizes a human bio-scaffold culture system. VBI will develop a market-ready 3D-liver assay platform adaptable to HTS applications. Sale of 3D HuBiogel culture kits or in-house bioassay services would have significant world-wide market, as a research & diagnostic tool. PUBLIC HEALTH RELEVANCE: Current cell-based assay models for studying drug metabolism and toxicity are of limited utility because isolated liver cells die rapidly in their no-physiologic, 2-dimensional (2D) culture formats. In Phase I, we have shown that a novel human 3-dimensional (3D or high- density) biomatrix scaffold- HuBiogel supports long-term cultivation, survival and functions of primary human liver cells. In Phase II, we will complete the development of new mini-liver bioassay platform by employing 3D HuBiogel scaffolds combined with rotary or perfusion culture techniques. High throughput utility of this robust 3D hepatotoxicity assay will greatly advance biochemical, metabolic and molecular analysis beyond 2D culture methods. Thus, our R&D effort provides a much needed in vitro tool for real-time analysis of chemical/drug metabolism and liver toxicity responses in humans. A successful outcome will offer new market-ready bioassay kits and services with world-wide demand in both basic and pharmaceutical research areas.